Sodium (Na) reabsorbing renal epithelial cells contain amiloride sensitive Na channels which are sequestered to the microvillar domain of the apical membrane. Epithelial Na channels (ENaCs) mediate entry of Na from the apical fluid during the first stage of electrogenic Na transport. Localization of ENaC to the apical membrane domain is essential for the vectoral transport of Na across these epithelia. Recent molecular characterization of ENaC affords the opportunity to identify associated proteins involved in establishing and maintaining ENaC within the apical membrane domain, such as peripheral membrane and membrane cytoskeletal proteins. Our long term goal is to understand the molecular interactions between ENaC and associated proteins which are involved in establishing and maintaining ENaC within the microvillar domain of the apical membrane of renal epithelia and how these interactions are regulated under both normal and pathophysiological conditions. In this application we propose to focus on interactions between ENaC and Apx (Apical protein Xenopus), a novel ENaC associated protein originally cloned from Xenopus. Recently mRNA for a human homolog of Apx has been identified in kidney. Based upon data presented in the Preliminary studies, we hypothesize that Apx interacts with ENaC and elements of the spectrin-based cytoskeleton and functions in sequestering and/or stabilizing ENaC within the microvillar domain of the apical membrane of Na reabsorbing renal epithelia. There are three Specific Aims: 1) To test the hypothesis that Apx interacts with ENaC and elements of the spectrin-based apical membrane cytoskeleton in Na+ reabsorbing renal epithelial cells. 2) To test the hypothesis that Apx sequester and/or stabilizes ENaC within the microvillar domain of the cell surface. 3) To test the hypothesis that an Apx homolog is expressed in Na reabsorbing renal epithelial of the mammalian kidney and that this homolog is associated with mammalian ENaC. It is expected that at the conclusion of the proposed study we will have determined the role of Apx and its mammalian homolog in sequestering and/or stabilizing ENaC within the microvillar domain of the apical membrane of Na reabsorbing renal epithelia. In addition, the results will enhance our understanding of the role ENaC associated proteins play in the regulation of this important ion channel. Furthermore, it is expected that the results obtained will facilitate our understanding of the organization of the apical membrane cytoskeleton and associated proteins in renal epithelial cells under normal physiologic as well as pathophysiologic conditions such as ischemia and polycystic kidney disease.